Molecular cloning of human D-dopachrome tautomerase cDNA: N-terminal proline is essential for enzyme activation.
D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5,6-quinone (D-dopachrome) into 5,6-dihydroxyindole. This protein has an amino acid sequence that is highly homologous with that of macrophage migration inhibitory factor (MIF), which has the potential to catalyze D-dopachrome to 5,6-dihydroxyindole-2-carboxylic acid and is an important cytokine for T-lymphocyte activation. We isolated and sequenced a 566 bp-long cDNA encoding human D-dopachrome tautomerase. The cDNA contains an open reading frame encoding 118 amino acids, including the initiator methionine. The amino acid sequence of the protein shares 80% homology with that of the rat enzyme. Northern blot analysis demonstrated that mRNA of D-dopachrome tautomerase is expressed in a large amount in the liver, and to lesser extent in other organs, including the heart, lung and pancreas. After purification of D-dopachrome tautomerase expressed in E. coli, we confirmed that the recombinant protein catalyzes the conversion of D-dopachrome to 5,6-dihydroxyindole. Its catalytic mechanism is not well understood. We found that the protein completely lost the enzyme activity when the N-terminal proline residue was replaced with alanine by site-directed mutagenesis. This fact suggests that the N-terminal proline is essential for the catalytic mechanism. Although the precise pathophysiological function of D-dopachrome tautomerase remains to be elucidated, the present results could contribute to further understanding of isomerase activity in relation to the immune response.
Nishihira
J
J
Central Research Institute, School of Medicine Hokkaido University, Sapporo, Japan. j_nisihi@med.hokudai.ac.jp
Fujinaga
M
M
Kuriyama
T
T
Suzuki
M
M
Sugimoto
H
H
Nakagawa
A
A
Tanaka
I
I
Sakai
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000502:physiology*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D045563:Indolequinones; D007211:Indoles / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D011392:Proline / Q000737:chemistry; D011809:Quinones / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D045563:Indolequinones; D007211:Indoles; D011809:Quinones; D012333:RNA, Messenger; C001123:dopachrome; C030692:5,6-dihydroxy-2-indolylcarboxylic acid; D011392:Proline; D019746:Intramolecular Oxidoreductases; C042501:dopachrome isomerase; C033871:5,6-dihydroxyindole